pQE-TriSystem Vector

The pQE-TriSystem vector contains CAG, T5, and p10 promoters that enable 6xHis-tagged protein expression in mammalian, E. coli, and baculovirus-infected insect cells, respectively (see figure pQE TriSystem). Preliminary studies can be carried out in bacterial expression systems, using the strong T5 promoter, which is recognized by E. coli polymerase, and allows efficient expression of proteins in any E. coli strain. If expression in mammalian or insect cells is required — to obtain post-translational modifications, for example — the same construct can be used without the need for time-consuming subcloning procedures.

QIAexpress pQE vectors combine a powerful phage T5 promoter (recognized by E. coli RNA polymerase) with a double lac operator repression module to provide tightly regulated, high-level expression of recombinant proteins in E. coli. Protein synthesis is effectively blocked in the presence of high levels of lac repressor and the stability of cytotoxic constructs is enhanced. The pQE vectors (see table and figure pQE Vectors) enable placement of the 6xHis tag at either the N- or C-terminus of the recombinant protein.

Elements present in QIAexpress pQE Vectors

Element	Description
1. Optimized promoter/operator element	Consists of the phage T5 promoter and two lac operator sequences, which increase the probability of lac repressor binding and ensure efficient repression of the powerful T5 promoter
2. Synthetic ribosomal binding site RBSII	For efficient translation
3. His-tag coding sequence	Either 5' or 3' to the polylinker cloning region
4. Translational stop codons	In all reading frames for convenient preparation of expression constructs
5. Two strong transcriptional terminators	t0 from phage lambda, and T1 from the rrnB operon of E. coli, to prevent read-through transcription and ensure stability of the expression construct
6. ColE1 origin of replicatio	From pBR322
7. beta-lactamase gene (bla)	Confers ampicillin resistance

Inserts encoding proteins of interest are cloned into appropriate constructs and transformed into a suitable E. coli strain for expression. Expression is induced by addition of IPTG. Vector pQE-TriSystem constructs can be transformed into E. coli, used as a shuttle vector for recombinant protein expression in insect cells, or transfected into mammalian cells.

The QIAexpress Expression System provides high-level expression of proteins suitable for
many applications, including:

• Purification of functional, conformationally active proteins
• Purification under denaturing conditions for antibody production
• Crystallization for determination of three-dimensional structure
• Assays involving protein-protein and protein-DNA interactions